Antibacterial agents: salinamide derivatives

ABSTRACT

The invention provides compounds of formula (I): 
     
       
         
         
             
             
         
       
     
     and salts thereof, wherein X and Y have any of the values defined herein. The compounds inhibit bacterial RNA polymerase, inhibit bacterial growth, and have applications in, analysis of RNA polymerase structure and function, control of bacterial gene expression, control of bacterial growth, antibacterial chemistry, antibacterial therapy, and drug discovery.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/104,914, filed Dec. 12, 2013, which claims the benefit of U.S.Provisional Application Ser. No. 61/736,476, filed on Dec. 12, 2012, nowexpired. The entire content of the applications referenced above arehereby incorporated by reference herein.

GOVERNMENT FUNDING

This invention was made with government support under grants AI072766,AI104660 and GM041376 awarded by the National Institutes of

Health. The government has certain rights in this invention.

BACKGROUND

Bacterial infectious diseases kill 100,000 persons each year in the USand 11 million persons each year worldwide, representing nearly a fifthof deaths each year worldwide (Heron et al., Final Data for 2006.National Vital Statistics Reports, Vol. 57 (Centers for Disease Controland Prevention, Atlanta Ga.) and World Health Organization (2008) TheGlobal Burden of Disease: 2004 Update (World Health Organization,Geneva)). In the US, hospital-acquired bacterial infections strike 2million persons each year, resulting in 90,000 deaths and an estimated$30 billion in medical costs (Klevins et al., (2007) Estimating healthcare-associated infections and deaths in U.S. hospitals. Public HealthReports, 122, 160-166; Scott, R. (2009) The direct medical costs ofhealthcare-associated infections in U.S. hospitals and benefits ofprevention (Centers for Disease Control and Prevention, Atlanta Ga.)).Worldwide, the bacterial infectious disease tuberculosis kills nearly 2million persons each year. One third of the world's population currentlyis infected with tuberculosis, and the World Health Organizationprojects that there will be nearly 1 billion new infections by 2020, 200million of which will result in serious illness, and 35 million of whichwill result in death.

For six decades, antibiotics have been a bulwark against bacterialinfectious diseases. This bulwark is failing due to the appearance ofresistant bacterial strains. For all major bacterial pathogens, strainsresistant to at least one current antibiotic have arisen. For severalbacterial pathogens, including tuberculosis, strains resistant to allcurrent antibiotics have arisen.

Bacterial RNA polymerase (RNAP) is a target for antibacterial therapy(Darst, S. (2004) Trends Biochem. Sci. 29, 159-162; Chopra, I. (2007)Curr. Opin. Investig. Drugs 8, 600-607; Villain-Guillot, P., Bastide,L., Gualtieri, M.

& Leonetti, J. (2007) Drug Discov. Today 12, 200-208; Ho, M., Hudson,B., Das, K., Arnold, E., Ebright, R. (2009) Curr. Opin. Struct. Biol.19, 715-723; and Srivastava et al. (2011) Curr. Opin. Microbiol. 14,532-543). The suitability of bacterial RNAP as a target forantibacterial therapy follows from the fact that bacterial RNAP is anessential enzyme (permitting efficacy), the fact that bacterial RNAPsubunit sequences are highly conserved (permitting broad-spectrumactivity), and the fact that bacterial RNAP-subunit sequences are nothighly conserved in human RNAP I, RNAP II, and RNAP III (permittingtherapeutic selectivity).

Accordingly, new antibacterial agents are needed.

SUMMARY OF CERTAIN EMBODIMENTS OF THE INVENTION

The invention provides new compositions of matter that inhibit bacterialRNA polymerase and inhibit bacterial growth. The compounds describedherein are anticipated to have applications in analysis of RNApolymerase structure and function, control of bacterial gene expression,control of bacterial growth, antibacterial chemistry, antibacterialtherapy, and drug discovery.

Accordingly, the invention provides a compound according to generalstructural formula (I):

wherein:

X is one of —Br, —I, —OR, —SR, and —NHR; Y is one of —Br, —I, —OR, —SR,and —NHR; and at least one of X and Y is OH;

each R is independently H or a branched or unbranched, saturated orunsaturated, hydrocarbon chain, having from 3 to 15 carbon atoms,wherein one or more of the carbon atoms is optionally replaced by (—O—)or (—NR^(a)—), and wherein the chain is optionally substituted on carbonwith one or more substituents independently selected from the groupconsisting of (C₁-C₆)alkoxy, (C₃-C₆)cycloalkyl, (C₁-C₆)alkanoyl,(C₁-C₆)alkanoyloxy, (C₁-C₆)alkoxycarbonyl, (C₁-C₆)alkylthio, azido,cyano, nitro, halo, hydroxy, oxo, carboxy, aryl, aryloxy, heteroaryl,heteroaryloxy, a hydrogen-bonding group, and a negatively chargedfunctional group; and

each R^(a) is independently H or (C₁-C₆)alkyl;

or a salt thereof.

The invention provides methods of structure-based design, synthesis, andassay of a compound according to general structural formula (I).

The invention provides use of a compound according to general structuralformula (I), e.g., to promote an anti-bacterial effect.

The invention also encompasses a crystal structure of a bacterial RNApolymerase in complex with salinamide A and a crystal structure of abacterial RNA polymerase in complex with a compound according to generalstructural formula (I).

The compounds of this invention have utility as RNAP inhibitors.

The compounds of this invention have utility as antibacterial agents.

The invention provides novel derivatives of salinamide A that containreplacements of the salinamide A epoxide that, it is believed, provideone or more of the following advantages as compared to the salinamide Aepoxide: (1) improvement of interactions with the salinamide bindingsite and an adjacent pocket on a bacterial RNA polymerase (e.g.,improving interactions with a residue corresponding to, and alignablewith, one of residues beta678, beta1105, beta1106, beta′731, andbeta′736 of Escherichia coli RNA polymerase), (2) increased potency ofinhibition of a bacterial RNA polymerase, (3) increased potency ofantibacterial activity, (4) increased stability, and (5) decreasedgenotoxicity.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Structures of SalA (compound 1) and SalB (compound 2).

FIGS. 2A-C. Crystal structure of RNAP in complex with Sal: overview.(FIG. 2A) Crystallization and refinement statistics for crystalstructure of Escherichia coli RNAP holoenzyme in complex with SalA at3.9 Å resolution. (FIG. 2B) Overall structure (two orthogonal views;gray surface labelled “*,” SalA; dark sphere, RNAP active-center Mg²⁺ion). (FIG. 2C) Electron density and model for SalA [two orthogonalviews; mesh, F₀-F_(C) omit map for SalA (NCS averaged and contoured at3.2σ); BH, bridge helix; FL, fork loop; LR, link region.

FIGS. 3A-B. Crystal structure of RNAP in complex with Sal: details.

(FIG. 3A) Stereoview showing RNAP-Sal interactions as observed in thecrystal structure of Escherichia coli RNAP holoenzyme in complex withSalA at 3.9 Å resolution. Gray, RNAP backbone (ribbon representation)and RNAP side-chain atoms (stick representation). Dashed lines, H-bonds.(FIG. 3B) Schematic summary of contacts between RNAP and SalA. Blackcircle, part of SalA that has unobstructed access to RNAP secondarychannel and RNAP active-center i+1 site. Dashed lines, H-bonds. Arcs,van der Waals interactions. .

FIGS. 4A-B. Crystal structure of RNAP in complex with Sal derivative.(FIG. 4A) Electron density, bromine anomalous difference density, andmodel for Escherichia coli RNAP holoenzyme in complex with Sal-Br (twoorthogonal views). Dark mesh, F₀-F_(C) omit map for SalA (NCS averagedand contoured at 3.2σ); light mesh labelled “Br”, bromine anomalousdifference density (contoured at 7σ); BH, bridge helix; FL, fork loop;LR, link region. (FIG. 4B) Schematic summary of contacts between RNAPand Sal-Br. Black circle, part of SalA that has unobstructed access toRNAP secondary channel and RNAP active-center i+1 site. Dashed lines,H-bonds. Arcs, van der Waals interactions.

DETAILED DESCRIPTION OF THE INVENTION

The following definitions are used, unless otherwise described: halo isfluoro, chloro, bromo, or iodo. Alkyl, alkoxy, alkenyl, alkynyl, etc.denote both straight and branched groups; but reference to an individualradical such as propyl embraces only the straight chain radical, abranched chain isomer such as isopropyl being specifically referred to.Aryl denotes a phenyl radical or an ortho-fused bicyclic carbocyclicradical having about nine to ten ring atoms in which at least one ringis aromatic. Heteroaryl encompasses a radical of a monocyclic aromaticring containing five or six ring atoms consisting of carbon and one tofour heteroatoms each selected from the group consisting of non-peroxideoxygen, sulfur, and N(X) wherein X is absent or is H, O, (C₁-C₄)alkyl,phenyl or benzyl, as well as a radical of an ortho-fused bicyclicheterocycle of about eight to ten ring atoms comprising one to fourheteroatoms each selected from the group consisting of non-peroxideoxygen, sulfur, and N(X). The following definitions are used, unlessotherwise indicated.

The term “hydrogen-bonding group” includes moieties that contain an O,N, or S atom able to donate or accept a hydrogen bond in aqueoussolution, such as, for example, amine, hydroxyl, thiol, ether,thioether, carbonyl, thionyl, carboxyl, thiocarboxyl, amide, thioamide,ester, thioester, sulfonic acid, sulfonic acid ester, sulfonamide,phosphoric acid, phosphoric acid ester, phosphonamide, boronic acid,boronic acid ester, pyrrole, pyrrolidine, carbazole, pyrroline, indole,isoindole, indoline, indolizine, furan, pyran, benzofuran, thiophene,benzothiophene, pyridine, quinoline, isoquinoline, quinazoline,napthyridine, oxazole, isoxazole, benzoxazole, thiazole, isothiazole,benthiazole, oxadiazole, thiadiazole, imidazole, triazole, tetrazole,benzimidazole, pyrazole, pyrazine, pyridazine, pyrimidine, triazine,indazole, purine, pteridine, phthalazine, quinoxaline, quinazoline,cinnoline, acridine, phenazine, phenothiazine, phenoxazine, and ionizedforms and salts thereof, as known to those skilled in the art.

The term “negatively charged functional group” includes moieties thatcontain an O, N, or S atom that predominantly carries a -1 negativecharge in aqueous solution at a physiologically relevant pH, betweenabout pH 4 and about pH 10, such as, for example, carboxyl,thiocarboxyl, sulfonic acid, phosphoric acid, phosphoric acid ester,boronic acid, triazole, tetrazole, purine, thiol, and ionized forms andsalts thereof, as known to those skilled in the art.

A combination of substituents or variables is permissible only if such acombination results in a stable or chemically feasible compound. Theterm “stable compounds,” as used herein, refers to compounds whichpossess stability sufficient to allow for their manufacture and whichmaintain the integrity of the compound for a sufficient period of timeto be useful for the purposes detailed herein (e.g., formulation intotherapeutic products, intermediates for use in production of therapeuticcompounds, isolatable or storable intermediate compounds, treating adisease or condition responsive to therapeutic agents).

Unless otherwise stated, structures depicted herein are also meant toinclude all stereochemical forms of the structure (i.e., the R and Sconfigurations for each asymmetric center). Therefore, singlestereochemical isomers, as well as enantiomeric and diastereomericmixtures, of the present compounds are within the scope of theinvention. Similarly, E- and Z-isomers, or mixtures thereof, of olefinswithin the structures also are within the scope of the invention.

When a bond in a compound formula herein is drawn in anon-stereochemical manner (e.g. flat), the atom to which the bond isattached includes all stereochemical possibilities. When a bond in acompound formula herein is drawn in a defined stereochemical manner(e.g. bold, bold-wedge, dashed or dashed-wedge), it is to be understoodthat the atom to which the stereochemical bond is attached is enrichedin the absolute stereoisomer depicted unless otherwise noted. In oneembodiment, the compound may be at least 51% the absolute stereoisomerdepicted. In another embodiment, the compound may be at least 60% theabsolute stereoisomer depicted. In another embodiment, the compound maybe at least 80% the absolute stereoisomer depicted. In anotherembodiment, the compound may be at least 90% the absolute stereoisomerdepicted. In another embodiment, the compound may be at least 95 theabsolute stereoisomer depicted. In another embodiment, the compound maybe at least 99% the absolute stereoisomer depicted.

Unless otherwise stated, structures depicted herein also are meant toinclude compounds that differ only by the presence of one or moreisotopically enriched atoms. For example, compounds having the presentstructures except for the replacement of a hydrogen atom by a deuteriumor tritium, or the replacement of a carbon by a ¹³C- or ¹⁴C-enrichedcarbon, are within the scope of this invention.

Compounds of this invention may exist in tautomeric forms, such asketo-enol tautomers. The depiction of a single tautomer is understood torepresent the compound in all of its tautomeric forms.

The term “pharmaceutically acceptable,” as used herein, refers to acomponent that is, within the scope of sound medical judgment, suitablefor use in contact with the tissues of humans and other mammals withoutundue toxicity, irritation, allergic response and the like, and arecommensurate with a reasonable benefit/risk ratio. A “pharmaceuticallyacceptable salt” means any non-toxic salt that, upon administration to arecipient, is capable of providing, either directly or indirectly, acompound of this invention. Accordingly, certain embodiments of theinvention are directed to salts of the compounds described herein, e.g.,pharmaceutically acceptable salts.

Acids commonly employed to form pharmaceutically acceptable saltsinclude inorganic acids such as hydrogen bisulfide, hydrochloric acid,hydrobromic acid, hydroiodic acid, sulfuric acid and phosphoric acid, aswell as organic acids such as para-toluenesulfonic acid, salicylic acid,tartaric acid, bitartaric acid, ascorbic acid, maleic acid, besylicacid, fumaric acid, gluconic acid, glucuronic acid, formic acid,glutamic acid, methanesulfonic acid, ethanesulfonic acid,benzenesulfonic acid, lactic acid, oxalic acid, para-bromophenylsulfonicacid, carbonic acid, succinic acid, citric acid, benzoic acid and aceticacid, as well as related inorganic and organic acids. Suchpharmaceutically acceptable salts thus include sulfate, pyrosulfate,bisulfate, sulfite, bisulfate, phosphate, monohydrogenphosphate,dihydrogenphosphate, metaphosphate, pyrophosphate, chloride, bromide,iodide, acetate, propionate, decanoate, caprylate, acrylate, formate,isobutyrate, caprate, heptanoate, propiolate, oxalate, malonate,succinate, suberate, sebacate, fumarate, maleate, butyne-1,4-dioate,hexyne-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate,dinitrobenzoate, hydroxybenzoate, methoxybenzoate, phthalate,terephthalate, sulfonate, xylene sulfonate, phenylacetate,phenylpropionate, phenylbutyrate, citrate, lactate, β-hydroxybutyrate,glycolate, maleate, tartrate, methanesulfonate, propanesulfonate,naphthalene-1-sulfonate, naphthalene-2-sulfonate, mandelate and othersalts. In one embodiment, pharmaceutically acceptable acid additionsalts include those formed with mineral acids such as hydrochloric acidand hydrobromic acid, and especially those formed with organic acidssuch as maleic acid.

The pharmaceutically acceptable salt may also be a salt of a compound ofthe present invention having an acidic functional group, such as acarboxylic acid functional group, and a base. Exemplary bases include,but are not limited to, hydroxide of alkali metals including sodium,potassium, and lithium; hydroxides of alkaline earth metals such ascalcium and magnesium; hydroxides of other metals, such as aluminum andzinc; ammonia, organic amines such as unsubstituted orhydroxyl-substituted mono-, di-, or tri-alkylamines, dicyclohexylamine;tributyl amine; pyridine; N-methyl, N-ethylamine; diethylamine;triethylamine; mono-, bis-, or tris-(2—OH-(C₁-C₆)-alkylamine), such asN,N-dimethyl-N-(2-hydroxyethyl)amine or tri-(2-hydroxyethyl)amine;N-methyl-D-glucamine; morpholine; thiomorpholine; piperidine;pyrrolidine; and amino acids such as arginine, lysine, and the like.

Antibacterial Agents

Specific values listed below for radicals, substituents, and ranges, arefor illustration only; they do not exclude other defined values or othervalues within defined ranges for the radicals and substituents

Specifically, (C₁-C₆)alkyl can be methyl, ethyl, propyl, isopropyl,butyl, iso-butyl, sec-butyl, pentyl, 3-pentyl, or hexyl;(C₃-C₆)cycloalkyl can be cyclopropyl, cyclobutyl, cyclopentyl, orcyclohexyl; (C₃-C₆)cycloalkyl can be cyclopropyl, cyclobutyl,cyclopentyl, or cyclohexyl; (C₁-C₆)alkoxy can be methoxy, ethoxy,propoxy, isopropoxy, butoxy, iso-butoxy, sec-butoxy, pentoxy, 3-pentoxy,or hexyloxy; (C₁-C₆)alkanoyl can be acetyl, propanoyl or butanoyl;(C₁-C₆)alkoxycarbonyl can be methoxycarbonyl, ethoxycarbonyl,propoxycarbonyl, isopropoxycarbonyl, butoxycarbonyl, pentoxycarbonyl, orhexyloxycarbonyl; (C₂-C₆)alkanoyloxy can be acetoxy, propanoyloxy,butanoyloxy, isobutanoyloxy, pentanoyloxy, or hexanoyloxy; aryl can bephenyl, indenyl, or naphthyl; and heteroaryl can be furyl, imidazolyl,triazolyl, triazinyl, oxazoyl, isoxazoyl, thiazolyl, isothiazoyl,pyrazolyl, pyrrolyl, pyrazinyl, tetrazolyl, pyridyl, (or its N-oxide),thienyl, pyrimidinyl (or its N-oxide), indolyl, isoquinolyl (or itsN-oxide) or quinolyl (or its N-oxide).

Certain embodiments of the present invention provide a compound ofgeneral structural formula (I):

wherein:X is one of —Br, —I, —OR, —SR, and —NHR; Y is one of —Br, —I, —OR, —SR,and —NHR; and at least one of X and Y is OH;

R is H or a branched or unbranched, saturated or unsaturated,hydrocarbon chain, having from 3 to 15 carbon atoms, wherein one or moreof the carbon atoms is optionally replaced by (—O—) or (—NR^(a)—), andwherein the chain is optionally substituted on carbon with one or moresubstituents independently selected from the group consisting of(C₁-C₆)alkoxy, (C₃-C₆)cycloalkyl, (C₁-C₆)alkanoyl, (C₁-C₆)alkanoyloxy,(C₁-C₆)alkoxycarbonyl, (C₁-C₆)alkylthio, azido, cyano, nitro, halo,hydroxy, oxo, carboxy, aryl, aryloxy, heteroaryl, heteroaryloxy, ahydrogen-bonding group, and a negatively charged functional group; and

each R^(a) is independently H or (C₁-C₆)alkyl; or a salt thereof.

In certain embodiments the compound of formula (I) is a compound offormula (Ia):

In certain embodiments R is a branched or unbranched, saturated orunsaturated, hydrocarbon chain, having from 3 to 8 carbon atoms, whereinone or more of the carbon atoms is optionally replaced by (—O—) or(—NR^(a)—), and wherein the chain is optionally substituted on carbonwith one or more substituents independently selected from the groupconsisting of (C₁-C₆)alkoxy, (C₁-C₆)alkanoyl, (C₁-C₆)alkanoyloxy,(C₁-C₆)alkoxycarbonyl, halo, hydroxy, oxo, carboxy, aryl, aryloxy, ahydrogen-bonding group, and a negatively charged functional group.

In certain embodiments, R consists of a chain of about 3 to about 6consecutively bonded non-hydrogen atoms and preferably contains ahydrogen-bonding or negatively charged functional group.

In certain embodiments, X is one of —Br and —I.

In certain embodiments, X is one of —OR,—SR, and —NHR, and R is H orconsists of a chain of about 3 to about 8 consecutively bondednon-hydrogen atoms and optionally contains one or more (e.g., 1, 2, or3) hydrogen-bonding or negatively charged functional groups.

In certain embodiments, X is one of —OR,—SR, and —NHR, and R is H orconsists of a chain of about 3 to about 8 consecutively bondednon-hydrogen atoms and contains one or more (e.g., 1, 2, or 3)hydrogen-bonding or negatively charged functional groups.

In certain embodiments, X is one of —OR,—SR, and —NHR, and R is H orconsists of a chain of about 3 to about 6 consecutively bondednon-hydrogen atoms and optionally contains one or more (e.g., 1, 2, or3) hydrogen-bonding or negatively charged functional groups.

In certain embodiments, X is one of —OR,—SR, and —NHR, and R is H orconsists of a chain of about 3 to about 6 consecutively bondednon-hydrogen atoms and contains one or more (e.g., 1, 2, or 3)hydrogen-bonding or negatively charged functional groups.

In certain embodiments, X is one of —O(CH₂)_(n)C(OH)(R′)R″,—O(CH₂)_(n)C(O)R′, —O(CH₂)_(n)C(O)OR′, —O(CH₂)_(n)C(O)NR′R″,—O(CH₂)_(n)OC(H)(R′)R″, —S(CH₂)_(n)C(OH)(R′)R″, —S(CH₂)_(n)C(O)R′,—S(CH₂)_(n)C(O)OR′, —S(CH₂)_(n)C(O)NR′R″, —S(CH₂)_(n)OC(H)(R′)R″,—NH(CH₂)_(n)C(OH)(R′)R″, —NH(CH₂)_(n)C(O)R′, —NH(CH₂)_(n)C(O)OR′,—NH(CH₂)_(n)C(O)NR′R″, and —NH(CH₂)_(n)OC(H)(R′)R″; wherein n is 1, 2,3, 4, 5, 6, or 7; and wherein R′ and R″ each independently is one of H,C₁-C₃alkyl, and C₁-C₃ alkyl substituted by one or more (e.g. 1, 2, or 3)halogen.

In certain embodiments, Y is OH.

In certain embodiments, Y is one of —Br and —I. In certain embodiments,Y is one of —OR,—SR, and —NHR, and R is H or consists of a chain ofabout 3 to about 8 consecutively bonded non-hydrogen atoms andoptionally contains one or more (e.g., 1, 2, or 3) hydrogen-bonding ornegatively charged functional groups.

In certain embodiments, Y is one of —OR,—SR, and —NHR, and R is H orconsists of a chain of about 3 to about 8 consecutively bondednon-hydrogen atoms and contains one or more (e.g., 1, 2, or 3)hydrogen-bonding or negatively charged functional groups.

In certain embodiments, Y is one of —OR,—SR, and —NHR, and R is H orconsists of a chain of about 3 to about 6 consecutively bondednon-hydrogen atoms and optionally contains one or more (e.g., 1, 2, or3) hydrogen-bonding or negatively charged functional groups.

In certain embodiments, Y is one of —OR,—SR, and —NHR, and R is H orconsists of a chain of about 3 to about 6 consecutively bondednon-hydrogen atoms and contains one or more (e.g., 1, 2, or 3)hydrogen-bonding or negatively charged functional groups.

In certain embodiments, Y is one of —O(CH₂)_(n)C(OH)(R′)R″,—O(CH₂)_(n)C(O)R′, —O(CH₂)_(n)C(O)OR′, —O(CH₂)_(n)C(O)NR′R″,—O(CH₂)_(n)OC(H)(R′)R″, —S(CH₂)_(n)C(OH)(R′)R″, —S(CH₂)_(n)C(O)R′,—S(CH₂)_(n)C(O)OR′, —S(CH₂)_(n)C(O)NR′R″, —S(CH₂)_(n)OC(H)(R′)R″,—NH(CH₂)_(n)C(OH)(R′)R″, —NH(CH₂)_(n)C(O)R′, —NH(CH₂)_(n)C(O)OR′,—NH(CH₂)_(n)C(O)NR′R″, and —NH(CH₂)_(n)OC(H)(R′)R″; wherein n is 1, 2,3, 4, 5, 6, or 7; and wherein R′ and R″ each independently is one of H,C₁-C₃alkyl, and C₁-C₃ alkyl substituted by one or more (e.g. 1, 2, or 3)halogen.

In certain embodiments, n is 1, 2, 3, 4, or 5.

Certain embodiments of the present invention provide a method ofstructure-based design of a compound described here that includesinspection of a crystal structure of a bacterial RNA polymerase incomplex with one of salinamide A and a compound described herein.

Certain embodiments of the present invention provide a method ofsynthesis of a compound described herein, comprising reaction ofsalinamide A with HX in the presence of an acid.

Certain embodiments of the present invention provide a method ofsynthesis of a compound described herein, comprising reaction ofsalinamide A with HX in the presence of a base.

Certain embodiments of the present invention provide a method ofsynthesis of a compound described herein, comprising reaction ofsalinamide A with YX, wherein Y is a cation.

Certain embodiments of the present invention provide a method ofsynthesis of a compound described herein, comprising reaction ofsalinamide B with HX in the presence of a base.

Certain embodiments of the present invention provide a method ofsynthesis of a compound described herein, comprising reaction ofsalinamide B with YX, wherein Y is a cation.

Certain embodiments of the present invention provide an assay forinhibition of a RNA polymerase comprising contacting a bacterial RNApolymerase with a compound described herein.

Certain embodiments of the present invention provide an assay forpotential antibacterial activity comprising contacting a bacterium witha compound described herein.

Certain embodiments of the present invention provide a use of a compounddescribed herein as an inhibitor of a bacterial RNA polymerase.

Certain embodiments of the present invention provide a use of a compounddescribed herein as an antibacterial agent.

Certain embodiments of the present invention provide a use of a compounddescribed herein as one of a disinfectant, a sterilant, an antispoilant,an antiseptic, and an antiinfective.

Certain embodiments of the present invention provide a pharmaceuticalcomposition comprising a compound of formula (I), or a pharmaceuticallyacceptable salt thereof, and a pharmaceutically acceptable carrier.

Certain embodiments of the present invention provide a compound offormula (I), or a pharmaceutically acceptable salt thereof, for use intherapy.

Certain embodiments of the present invention provide a method to treat abacterial infection in an animal (e.g. a mammal, such as a human)comprising administering a compound of formula (I), or apharmaceutically acceptable salt thereof, to the animal. The inventionalso provides a synthetic intermediate of formula 7 or 9:

or a salt thereof. The synthetic intermediates are useful for preparingother compounds of formula (I).

Applicant has synthesized the compound according to general structuralformula (I) wherein X is bromine.

Applicant has shown that the compound according to general structuralformula (I) wherein X is bromine potently inhibits bacterial RNApolymerase (RNAP) in vitro (Escherichia coli RNAP; IC50=0.78±0.05 μM[radiochemical assay]; Staphylococcus aureus RNAP; IC50=0.54±0.04 μM[radiochemical assay]), and does not detectably inhibit human RNAP I, H,and III (IC50>100 μM [radiochemical assay])

Applicant has shown that the compound according to general structuralformula (I) wherein X is bromine exhibits potent antibacterial activityagainst Gram-negative bacteria in culture (Escherichia coli D21f2tolC,MIC50=0.049 μg/ml; Enterobacter cloacae, MIC50=1.56 μg/ml; Neisseriagonorrhoeae, MIC50=1.56 μg/ml; Haemophilus influenzae, MIC50=6.25 μg/ml;Pseudomonas aeruginosa, MIC50=50 μg/ml), and does not detectably inhibitgrowth of mammalian cells in culture (Vero E6 cells, MIC>50 μg/ml).

Applicant has synthesized the compounds according to general structuralformula (I) wherein X is —OH, —OBu, —NH(CH₂)₃NHBoc, and —NH(CH₂)₃NHBoc).

Applicant has shown that the compounds according to general structuralformula (I) wherein wherein X is —OH, —OBu, —NH(CH₂)₃NHBoc, or—NH(CH₂)₃NHBoc potently inhibit bacterial RNAP in vitro (Escherichiacoli RNAP; IC50s=0.3-6 μM [fluorescence-detected assays]; Table 1).

Applicant has shown that the compounds according to general structuralformula (I) wherein X is —OH, —OBu, —NH(CH₂)₃NHBoc, or —NH(CH₂)₃NHBocexhibit potent antibacterial activity against Gram-negative bacteria inculture (Escherichia coli D21f2tolC, MIC50s=0.78-1.56 μg/ml;Enterobacter cloacae, MIC50s=12.5.-100 μg/ml; Table 2). Applicant hasdetermined crystal structures of (1) Escherichia coli RNAP in complexwith salinamide A and (2) Escherichia coli RNAP in complex with thecompound according to general structural formula (I) wherein X isbromine. The crystal structures enable structure-based design ofcompounds according to general structural formula (I).

Salinamides

Compounds according to general structural formula (I) are analogs ofsalinamide A (Sal; SalA; compound 1) and salinamide B (SalB; compound2).

SalA and SalB are bicyclic depsipeptides, each consisting of sevenamino-acid residues and two non-amino-acid residues (Trischman et al.,J. Am. Chem. Soc., 116:757-758, 1994; Moore et al., J. Org. Chem.,64:1145-1150, 1999; FIG. 1). Residue 9 of SalA contains an epoxidemoiety. Residue 9 of SalB contains a chlorohydrin moiety.

SalA and SalB are produced by Streptomyces sp. CNB-091, a marinebacterium isolated from the surface of the jellyfish Cassiopeiaxamachana (Trischman et al., J. Am. Chem. Soc., 116:757-758, 1994; Mooreet al., J. Org. Chem., 64:1145-1150, 1999; Moore & Seng, TetrahedronLett. 39:3915-3918, 1998). SalA also is produced by Streptomyces sp.NRRL 21611, a soil bacterium (Miao et al., J. Nat. Prod. 60, 858-861,1997).

A total synthesis of SalA has been reported (Tan & Ma, Angew. Chem. Int.Ed. 47:3614-3617, 2008).

Salinamides: RNAP-Inhibitory Activity and Antibacterial Activity

It has been reported previously that SalA inhibits Gram-positive andGram-negative bacterial RNAP in vitro (Miao et al., J. Nat. Prod. 60,858-861, 1997). It is disclosed herein that SalB also inhibitsGram-positive and Gram-negative bacterial RNAP in vitro. It further isdisclosed herein that SalA and SalB do not detectably inhibit human RNAPI, II, and III.

It has been reported previously that SalA and SalB exhibit antibacterialactivity against Gram-positive bacterial pathogens (Trischman et al., J.Am. Chem. Soc., 116:757-758, 1994; Moore et al., J. Org. Chem.,64:1145-1150, 1999). It is disclosed herein that SalA and SalB exhibitantibacterial activity against Gram-negative bacterial pathogens,including Enterobacter cloacae, Haemophilus influenzae, Neisseriagonorrhoeae, and Pseudomonas aeruginosa. It further is disclosed hereinthat SalA and SalB do not detectably inhibit growth of mammalian cellsin culture.

The inhibition of bacterial RNAP by Sal accounts, in part or in whole,for the antibacterial activity of Sal (Ebright et al., WO/2012/129173,2012). Sal inhibits RNA synthesis not only in vitro but also inbacterial cells in culture (Ebright et al., WO/2012/129173, 2012).Mutations in genes encoding RNAP beta and beta' subunits conferresistance to the antibacterial activity of Sal (Ebright et al.,WO/2012/129173, 2012).

Salinamides: Binding Site on RNAP

The binding site on bacterial RNAP for Sal—the“Sal target” (alsoreferred to as the “bridge-helix-cap target”)—was identified by mappingsites of substitutions that confer Sal-resistance onto thethree-dimensional structure of

RNAP (Ebright et al., WO/2012/129173, 2012).

The binding site on bacterial RNAP for Sal was confirmed by determiningcrystal structures of Escherichia coli RNAP holoenzyme in the absence ofSal (resolution=4.0 Å) and Escherichia coli RNAP holoenzyme in thepresence of Sal (resolution=4.2 Å) (Ebright et al., WO/2012/129173,2012). Comparison of electron density maps revealed difference densityattributable to Sal. The difference density was located in the Saltarget and was in contact with or close to sites of substitutionsconferring Sal resistance are obtained. The resolution was sufficient toconclude that the Sal target is the binding site on RNAP for Sal, andthat sites of substitutions that confer Sal-resistance correspond toRNAP residues of RNAP that contact or are close to Sal. However, theresolution was insufficient to define the orientation of Sal relative tothe Sal target and to define interatomic contacts between Sal and theSal target.

Disclosed herein are crystal structures of Escherichia coli RNAPholoenzyme in the absence of Sal and Escherichia coli RNAP holoenzyme inthe presence of Sal at a resolution sufficient to define the orientationof Sal relative to the Sal target and to define interatomic contactsbetween Sal and the Sal target (resolution, =3.9 Å; FIGS. 2A-C and3A-B).

Further disclosed herein are electron density and bromine anomalousdifference density for Escherichia coli RNAP holoenzyme in complex withSal-Br, the compound according to general structural formula (I) whereinX is bromine (FIGS. 4A-B). The location of the Sal-Br bromine anomalousdifference density peak relative to the Sal target unequivocallyconfirms the orientation of Sal relative to the Sal target (FIGS. 4A-B).

The Sal target is located adjacent to, and partly overlaps, the RNAPpolymerase active center (Ebright et al., WO/2012/129173, 2012). It isinferred that Sal most likely inhibits RNAP by inhibiting RNAPactive-center function.

The Sal target does not overlap the RNAP active-center Mg²⁺ion and doesnot overlap RNAP residues that interact with the DNA template, the RNAproduct, or the nucleoside triphosphate substrate (Ebright et al.,WO/2012/129173, 2012). It is inferred Sal inhibits RNAP active-centerfunction allosterically, through effects on RNAP conformation, ratherthan through direct interactions with RNAP residues that mediate bondformation, product binding, and substrate binding.

The Sal target overlaps an RNAP active-center module referred to as the“bridge-helix cap,” which, in turn, comprises three active-centersubregions: the “bridge-helix N-terminal hinge” (BH-H_(N)), the“F-loop,” and the “link region” (Ebright et al., WO/2012/129173, 2012).It has been proposed that the BH-H_(N) undergoeshinge-opening/hinge-closing conformational changes coupled to, andessential for, the nucleotide-addition cycle in RNA synthesis, and thatthe F-loop and link region, coordinate these conformational changes(Weinzierl, BMC Biol. 8:134, 2010; Hein & Landick, BMC Biol. 8:141,2010; Kireeva et al., BMC Biophys. 5:11-18, 2012; Nedialkov et al.,Biochim. Biophys. Acta 1829:187-198, 2013). It is inferred that Sal mayinhibit RNAP active-center function by inhibiting BH-H_(N) hinge-openingand/or hinge-closing (Ebright et al., WO/2012/129173, 2012).

The Sal target is located close to, but does not overlap, the target ofthe rifamycin antibacterial agents (e.g., rifampin, rifapentine,rifabutin, and rifalazil), which are RNAP inhibitors in current clinicaluse in antibacterial therapy (Ebright et al., WO/2012/129173, 2012; seeDarst.Trends Biochem. Sci. 29:159-162, 2004; Chopra, Curr. Opin.Investig. Drugs 8:600-607, 2007; Villain-Guillot et al., Drug Discov.Today 12:200-208, 2007; Ho et al., Curr. Opin. Struct. Biol. 19:715-723,2009). Consistent with the lack of overlap between the Sal target andthe rifamycin target, Sal-resistant mutants are not cross-resistant torifamycins, and rifamycin-resistant mutants are not cross-resistant toSal (Ebright et al., WO/2012/129173, 2012).

The Sal target also is located close to, but does not overlap, thetarget of CBR703, an RNAP inhibitor under investigation for clinical usein antibacterial therapy (Ebright et al., WO/2012/129173, 2012; seeDarst.Trends Biochem. Sci. 29:159-162, 2004; Chopra, Curr. Opin.Investig. Drugs 8:600-607, 2007; Villain-Guillot et al., Drug Discov.Today 12:200-208, 2007). Consistent with the lack of overlap between theSal target and the CBR703 target, Sal-resistant mutants are notcross-resistant to CBR703, and CBR703-resistant mutants are notcross-resistant to Sal (Ebright et al., WO/2012/129173, 2012).

It is disclosed herein that the Sal target does not overlap the targetsof the RNAP inhibitors streptolydigin, myxopyronin, and lipiarmycin (seeChopra, Curr. Opin. Investig. Drugs 8:600-607, 2007; Villain-Guillot etal., Drug Discov. Today 12:200-208, 2007; Ho et al., Curr. Opin. Struct.Biol. 19:715-723, 2009; Srivastava et al., Curr. Opin. Microbiol.14:532-543, 2011). The Sal target is located adjacent to, but does notoverlap, the streptolydigin target. The Sal target is distant from themyxopyronin and lipiarmycin targets. It further is disclosed hereinthat, consistent with the absence of overlap between the Sal target andthe streptolydigin, myxopyronin, and lipiarmycin targets, Sal-resistantmutants do not exhibit cross-resistance with streptolydigin,myxopyronin, and lipiarmycin, and, conversely, streptolydigin-resistant,myxopyronin-resistant, and lipiarmycin-resistant mutants do not exhibitcross-resistance with Sal.

Salinamides: Mechanism of Inhibition of RNAP

It is disclosed herein that Sal inhibits RNAP through a mechanism thatis different from the mechanisms of rifamycins, streptolydigin,myxopyronin, and lipiarmycin.

It is disclosed herein that Sal does not inhibit formation of theRNAP-promoter open complex in transcription initiation. This resultindicates that Sal inhibits RNAP through a mechanism different from themechanisms of myxopyronin and lipiarmycin (which inhibit formation ofRNAP-promoter open complex).

It is disclosed herein that Sal inhibits nucleotide addition in bothtranscription initiation and transcription elongation. Sal inhibits bothprimer-dependent transcription initiation and de novo transcriptioninitiation. In primer-dependent transcription initiation, Sal inhibitsall nucleotide-addition steps, including the first nucleotide-additionstep yielding a 3-nucleotide RNA product from a 2-nucleotide RNA primerand an NTP. In de novo transcription initiation, Sal inhibits allnucleotide-addition steps, including the first nucleotide-addition stepyielding a 2-nucleotide RNA product from two NTPs. These results confirmthat Sal inhibits RNAP through a mechanism different from the mechanismsof myxopyronin and lipiarmycin (which do not inhibit transcriptionelongation) and indicate that Sal inhibits RNAP through a mechanismdifferent from the mechanism of rifamycins (which do not inhibit thefirst nucleotide addition step in transcription initiation and which donot inhibit transcription elongation).

It is disclosed herein that transcription inhibition by Sal does notrequire the RNAP active-center subregion referred to as the triggerloop. Sal inhibits wild-type RNAP and an RNAP-derivative having adeletion of the trigger loop to equal extents and with nearly equalconcentration-dependences.. This result indicates that Sal inhibits RNAPthrough a mechanism different from the mechanisms of streptolydigin (forwhich transcription inhibition requires the trigger loop).

It is disclosed herein that transcription inhibition by Sal isnoncompetitive with respect to NTP substrate. It is inferred that Saldoes not inhibit the NTP binding sub-reaction of the nucleotide-additioncycle, but, instead, inhibits one or more of the bond-formation,pyrophosphate-release, and translocation sub-reactions of thenucleotide-addition cycle.

Salinamides: Novel Sal Analogs

The syntheses disclosed herein of Sal-Br, Sal-OH, Sal-OR, Sal-SR, andSal-NHR show that the SalA epoxide moiety and SalB chlorohydrin moietiesprovide chemical reactivity that can be exploited for semi-synthesis ofnovel Sal analogs (Examples 3-7). The observation that certainsynthesized Sal analogs retain RNAP-inhibitory activity andantibacterial activity shows that certain substitutions of the SalAepoxide moiety and SalB chlorohydrin moiety can be tolerated withoutloss of activity (Tables 1-2). The crystal structure of RNAP-Salindicates that the SalA epoxide moiety and SalB chlorohydrin moiety makefew or no interactions with RNAP and are located at the entrance to theSal binding pocket, directed towards the RNAP secondary channel and theRNAP active-center i+1 site (FIGS. 3A-B and 4A-B).

These findings set the stage for structure-based design ofsemi-synthetic, novel Sal analogs with increased potency.

By way of example, appending a sidechain that carries hydrogen-bondingfunctionality at the SalA epoxide moiety or SalB chlorohydrin moiety,could provide favorable hydrogen-bonded interactions with polar residueson the floor of the RNAP secondary channel (e.g., residues β678, β31105,β1106, β′731, and β′736 in RNAP from Escherichia coli, and residuescorresponding to, and alignable with, these residues in RNAP from otherbacterial species).

By further way of example, appending a sidechain carrying negativelycharged functionality at the SalA epoxide moiety or SalB chlorohydrinmoiety could provide favorable electrostatic interactions withpositively charged residues on the floor of the RNAP secondary channel(e.g., residues β678, β31106, and β′731 in RNAP from Escherichia coli,and residues corresponding to, and alignable with, these residues inRNAP from other bacterial species).

Administration of Pharmaceutical Compositions

The compounds described herein may be formulated as pharmaceuticalcompositions and administered to a mammalian host, such as a human maleor female patient in a variety of forms adapted to the chosen route ofadministration (e.g., orally or parenterally, by intravenous,intramuscular, topical or subcutaneous routes).

Thus, the present compounds may be systemically administered, e.g.,orally, in combination with a pharmaceutically acceptable vehicle suchas an inert diluent or an assimilable edible carrier. They may beenclosed in hard or soft shell gelatin capsules, may be compressed intotablets, or may be incorporated directly with the food of the patient'sdiet. For oral therapeutic administration, the active compound may becombined with one or more excipients and used in the form of ingestibletablets, buccal tablets, troches, capsules, elixirs, suspensions,syrups, wafers, and the like. Such compositions and preparations shouldcontain at least 0.1% of active compound. The percentage of thecompositions and preparations may, of course, be varied and mayconveniently be between about 2 to about 60% of the weight of a givenunit dosage form. The amount of active compound in such therapeuticallyuseful compositions is such that an effective dosage level will beobtained.

The tablets, troches, pills, capsules, and the like may also contain thefollowing: binders such as gum tragacanth, acacia, corn starch orgelatin; excipients such as dicalcium phosphate; a disintegrating agentsuch as corn starch, potato starch, alginic acid and the like; alubricant such as magnesium stearate; and a sweetening agent such assucrose, fructose, lactose or aspartame or a flavoring agent such aspeppermint, oil of wintergreen, or cherry flavoring may be added. Whenthe unit dosage form is a capsule, it may contain, in addition tomaterials of the above type, a liquid carrier, such as a vegetable oilor a polyethylene glycol. Various other materials may be present ascoatings or to otherwise modify the physical form of the solid unitdosage form. For instance, tablets, pills, or capsules may be coatedwith gelatin, wax, shellac or sugar and the like. A syrup or elixir maycontain the active compound, sucrose or fructose as a sweetening agent,methyl and propylparabens as preservatives, a dye and flavoring such ascherry or orange flavor. Of course, any material used in preparing anyunit dosage form should be pharmaceutically acceptable and substantiallynon-toxic in the amounts employed. In addition, the active compound maybe incorporated into sustained-release preparations and devices.

The active compound may also be administered intravenously orintraperitoneally by infusion or injection. Solutions of the activecompound or its salts can be prepared in water, optionally mixed with anontoxic surfactant. Dispersions can also be prepared in glycerol,liquid polyethylene glycols, triacetin, and mixtures thereof and inoils. Under ordinary conditions of storage and use, these preparationscontain a preservative to prevent the growth of microorganisms.

The pharmaceutical dosage forms suitable for injection or infusion caninclude sterile aqueous solutions or dispersions or sterile powderscomprising the active ingredient which are adapted for theextemporaneous preparation of sterile injectable or infusible solutionsor dispersions, optionally encapsulated in liposomes. In all cases, theultimate dosage form should be sterile, fluid and stable under theconditions of manufacture and storage. The liquid carrier or vehicle canbe a solvent or liquid dispersion medium comprising, for example, water,ethanol, a polyol (for example, glycerol, propylene glycol, liquidpolyethylene glycols, and the like), vegetable oils, nontoxic glycerylesters, and suitable mixtures thereof. The proper fluidity can bemaintained, for example, by the formation of liposomes, by themaintenance of the required particle size in the case of dispersions orby the use of surfactants. The prevention of the action ofmicroorganisms can be brought about by various antibacterial andantifungal agents, for example, parabens, chlorobutanol, phenol, sorbicacid, thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars, buffers or sodiumchloride. Prolonged absorption of the injectable compositions can bebrought about by the use in the compositions of agents delayingabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the activecompound in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfilter sterilization. In the case of sterile powders for the preparationof sterile injectable solutions, the preferred methods of preparationare vacuum drying and the freeze drying techniques, which yield a powderof the active ingredient plus any additional desired ingredient presentin the previously sterile-filtered solutions.

For topical administration, the present compounds may be applied in pureform, e.g., when they are liquids. However, it will generally bedesirable to administer them to the skin as compositions orformulations, in combination with a dermatologically acceptable carrier,which may be a solid or a liquid.

Useful solid carriers include finely divided solids such as talc, clay,microcrystalline cellulose, silica, alumina and the like. Useful liquidcarriers include water, alcohols or glycols or water-alcohol/glycolblends, in which the present compounds can be dissolved or dispersed ateffective levels, optionally with the aid of non-toxic surfactants.Adjuvants such as fragrances and additional antimicrobial agents can beadded to optimize the properties for a given use. The resultant liquidcompositions can be applied from absorbent pads, used to impregnatebandages and other dressings, or sprayed onto the affected area usingpump-type or aerosol sprayers.

Thickeners such as synthetic polymers, fatty acids, fatty acid salts andesters, fatty alcohols, modified celluloses or modified mineralmaterials can also be employed with liquid carriers to form spreadablepastes, gels, ointments, soaps, and the like, for application directlyto the skin of the user.

Examples of useful dermatological compositions which can be used todeliver the compounds of formula I to the skin are known to the art; forexample, see Jacquet et al. (U.S. Pat. No. 4,608,392), Geria (U.S. Pat.No. 4,992,478), Smith et al. (U.S. Pat. No. 4,559,157) and Wortzman(U.S. Pat. No. 4,820,508).

Useful dosages of the compounds of formula I can be determined bycomparing their in vitro activity, and in vivo activity in animalmodels. Methods for the extrapolation of effective dosages in mice, andother animals, to humans are known to the art; for example, see U.S.Pat. No. 4,938,949.

The amount of the compound, or an active salt or derivative thereof,required for use in treatment will vary not only with the particularsalt selected but also with the route of administration, the nature ofthe condition being treated and the age and condition of the patient andwill be ultimately at the discretion of the attendant physician orclinician.

In general, however, a suitable dose will be in the range of from about0.5 to about 150 mg/kg, e.g., from about 10 to about 125 mg/kg of bodyweight per day, such as 3 to about 75 mg per kilogram body weight of therecipient per day, preferably in the range of 6 to 120 mg/kg/day, mostpreferably in the range of 15 to 90 mg/kg/day.

The compound may be conveniently formulated in unit dosage form; forexample, containing 5 to 1000 mg, conveniently 10 to 750 mg, mostconveniently, 50 to 500 mg of active ingredient per unit dosage form. Inone embodiment, the invention provides a composition comprising acompound of the invention formulated in such a unit dosage form.

The desired dose may conveniently be presented in a single dose or asdivided doses administered at appropriate intervals, for example, astwo, three, four or more sub-doses per day. The sub-dose itself may befurther divided, e.g., into a number of discrete loosely spacedadministrations; such as multiple inhalations from an insufflator or byapplication of a plurality of drops into the eye.

The invention will now be illustrated by the following non-limitingExamples.

EXAMPLES Example 1 Crystal Structure of RNAP in Complex with Sal

Crystal structures of Escherichia coli RNAP holoenzyme at 3.9 Åresolution and Escherichia coli RNAP holoenzyme in complex with SalA at3.9 Å resolution were determined as follows:

Crystallization trials were performed using Crystal Former microfluidicchips (Microlytic, Inc.) and SmartScreen solutions (Microlytic, Inc.)(precipitant inlet: 1.5 μl screening solution; sample inlet: 1.5 μl 10mg/ml Escherichia coli RNAP holoenzyme in 10 mM Tris-HCl, pH 7.9, 100 mMNaCl, 1% glycerol; 22° C.). Under one condition, small crystals appearedwithin two days. Conditions were optimized using the hanging-dropvapor-diffusion technique at 22° C. The optimized conditions (reservoir:500 μl 0.1 M HEPES, pH 7.0, 0.2 M CaCl₂, and 18% PEG400; drop: 1 μl 10mg/ml Escherichia coli RNAP holoenzyme in 10 mM Tris-HCl, pH 7.9, 100 mMNaCl, 1% glycerol plus 1 μl reservoir solution; 22° C.) yielded largecrystals with dimensions of 0.2 mm×0.2 mm×0.2 mm in one week. SalA wassoaked into RNAP crystals, yielding RNAP-Sal crystals, by addition of0.2 μl 20 mM SalA or Sal-Br in (±)-2-methyl-2,4-pentanediol (HamptonResearch, Inc.) to the crystallization drop and incubation 30 min at 22°C. RNAP and RNAP-SalA crystals were transferred to reservoir solutionscontaining 15% (v/v) (2R, 3R)-(−)-2, 3-butanediol (Aldrich, Inc.) andthen flash-cooled with liquid nitrogen.

Diffraction data were collected from cryo-cooled crystals at CornellHigh

Energy Synchrotron Source beamline F1 and at Brookhaven NationalLaboratory beamline X25. Data were processed using HKL2000.

The structure of Escherichia coli RNAP holoenzyme was solved bymolecular replacement using AutoMR. The search model was generated bystarting with the crystal structure of Thermus thermophilusRNAP-promoter open complex (PDB 4G7H), deleting DNA and non-conservedprotein domains, modelling Escherichia coli RNAP holoenzyme α^(I) andα^(II) subunit N-terminal domains by superimposing the crystal structureof Escherichia coli RNAP holoenzyme α N-terminal domain dimer (PRB1BDF), and modelling Escherichia coli RNAP holoenzyme β, β′, ω, and σ⁷⁰subunits using Sculptor (backbone and sidechain atoms for identicalresidues; backbone and Cβ atoms for non-identical residues). Two RNAPmolecules were present in the asymmetric unit. Crystal structures ofEscherichia coli RNAP holoenzyme α subunit C-terminal domain (PDB 3K4G),the Escherichia coli RNAP holoenzyme β subunit β2-βi4 and βflap-βi9domains (PDB 3LTI and PDB 3LU0), and Escherichia coli RNAP holoenzymeσ⁷⁰ region 2 (PDB 1 SIG) were fitted manually to the (Fo-Fc) differenceelectron density map. Early-stage refinement of the structure wasperformed using Phenix and included rigid-body refinement of each RNAPmolecule in the asymmetric unit, followed by rigid-body refinement ofeach subunit of each RNAP molecule, followed by rigid-body refinement of216 segments of each RNAP molecule, followed by group B-factorrefinement with one B-factor group per residue, using Phenix. Densitymodification, including non-crystallographic-symmetry averaging andsolvent flattening, were performed to remove model bias and to improvephases. The resulting maps allowed segments that were not present in thesearch model to be built manually using Coot. Cycles of iterative modelbuilding with Coot and refinement with Phenix improved the model. Thefinal E. coli RNAP holoenzyme model, refined to Rwork and Rfree of 0.276and 0.325, respectively, was deposited in the PDB with accession code4MEY.

The structure of Escherichia coli RNAP holoenzyme coli in complex withSalA was solved by molecular replacement in AutoMR, using the abovecrystal structure of Escherichia coli RNAP holoenzyme as the searchmodel. After rigid-body refinement with 216 domains, an electron densityfeature corresponding to one molecule of SalA per holoenzyme was clearlyvisible in the (Fo-Fc) difference map. A structural model of SalAderived from the crystal structure of SalB (CSD 50962; enantiomorphcorrected based on Moore et al., et al., J. Org. Chem., 64:1145-1150,1999) was fitted to the (Fo-Fc) difference map with minor adjustments ofSalA conformation. The final Escherichia coli RNAP holoenzyme-SalAcomplex model, refined to Rwork and Rfree of 0.286 and 0.325,respectively, was deposited in the PDB with accession code 4MEX.

The structure of E. coli RNAP holoenzyme in complex with Sal at 3.9 Åresolution shows unambiguous experimental electron density for Sal inthe genetically-defined Sal target, confirming the hypothesis that theSal target represents the Sal binding site on RNAP (FIGS. 2A-C).

The structure shows that Sal binds within the RNAP bridge-helix cap,making direct interactions with the BH-H_(N), the fork loop, and thelink region (FIGS. 2A-C and 3A-B).

Sal makes direct interactions with all residues at which substitutionsconferring highest-level (≧128-fold) Sal-resistance are obtained (β′residues R738, A779, and G782, and β residues D675 and N677; FIGS.3A-B).

Six residues that make direct contact with SalA are conserved acrossGram-positive bacterial RNAP, Gram-negative bacterial RNAP, and humanRNAP. Eight residues that contact Sal are conserved in Gram-positivebacterial RNAP and Gram-negative bacterial RNAP, but are not conserved,and indeed are radically different, in human RNAP. The observedinteractions account for and explain the observation that Sal inhibitsGram-positive and Gram-negative bacterial RNAP, but does not inhibithuman RNAP.

Sal binds within a ˜600 Å³ pocket formed by the BH-H_(N), the fork loop,and the link region. Backbone atoms of residues that form the pockethave the same conformations in RNAP holoenzyme in the absence of Sal andin RNAP holoenzyme in complex with Sal, indicating that the pocketpre-exists in RNAP holoenzyme in the absence of Sal.

The pocket opens at one end onto the RNAP secondary channel and the RNAPactive-center “i+1” NTP-insertion site. It seems likely that Sal entersthe pocket from the RNAP secondary channel or the active-center “i+1”site.

Within the binding pocket, Sal residues 4, 5, 7, and 8 interact with theRNAP BH-H_(N), Sal residues 1-3 and 6-7 interact with the RNAP forkloop, and Sal residues 8 and 9 interact with the RNAP link region (FIGS.3A-B). Sal residue 9 is at the end of the pocket that opens onto theRNAP secondary channel and the active-center “i+1” site (FIGS. 3A-B).The Sal residue-9 epoxide moiety and methyl moiety extend into thisopening and make only limited interactions with residues of RNAP (FIGS.3A-B).

The crystal structure of the RNAP-Sal complex also defines effects ofSal on RNAP conformation.

The crystal structure of RNAP-Sal shows that Sal interacts with theBH-H_(N) in an “open” (unbent) state. This conformation is differentfrom the “closed” (bent) BH-H_(N) conformation that has been observed inmolecular-dynamics simulations of nucleotide-addition reactions intranscription elongation complexes, and that has been postulated toserve as an intermediate in the pyrophosphate-release and/ortranslocation reactions of the nucleotide-addition cycle (Weinzierl, BMCBiol. 8:134, 2010; Hein & Landick, BMC Biol. 8:141, 2010; Kireeva etal., BMC Biophys. 5:11-18, 2012; Nedialkov et al., Biochim. Biophys.Acta 1829:187-198, 2013). It is inferred that Sal interacts with an“open” (unbent) BH-H_(N) conformational state, and it is hypothesizedthat, through its interactions with that state, Sal stabilizes thatstate and inhibits BH-H_(N) conformational dynamics required fornucleotide addition.

In the crystal structure of RNAP-Sal, the RNAP active-center triggerloop is disordered. Modeling indicates that the structure of RNAP-Salcould accommodate the “open” (unfolded) trigger loop conformationsobserved in crystal structures of some transcription initiation andelongation complexes, but could not accommodate the “closed” (folded)trigger loop conformations observed in other crystal structure oftranscription initiation and elongation complexes. It is inferred thatSal favors “open” (unfolded) trigger loop conformational states, and maydisfavor the “closed” (folded) trigger loop conformational states.However, experiments with an RNAP derivative lacking the trigger loopindicate that the trigger loop is not essential for transcriptioninhibition by Sal. Therefore, although effects of Sal on trigger loopconformation could contribute to transcription inhibition by Sal, theyare neither necessary nor sufficient for transcription inhibition bySal.

The interactions observed in the structure, or predicted based on thestructure, suggest opportunities for preparation of novel Sal analogswith improved potencies by use of semi-synthesis or by total synthesis.

The structure shows that the SalA residue-9 epoxide moiety is directedtoward the RNAP secondary channel and RNAP active-center “i+1” site(FIGS. 3A-B) but makes limited interactions with RNAP (FIGS. 3A-B). TheSalA epoxide can be altered with little or no loss of activity (Tables1-2), and has unique chemical reactivity (Examples 3-5). Accordingly, itis inferrd herein that it should be possible—by semi-synthesis or bytotal synthesis—to append at the SalA residue-9 epoxide moiety bychemical functionality that makes favorable interactions with the RNAPsecondary channel or active-center “i+1” site, thereby increasing thepotency of RNAP inhibitory activity and potentially increasing thepotency of antibacterial activity.

The structure predicts that the SalB residue-9 chlorohydrin moietylikewise makes limited interactions with RNAP and is directed toward theRNAP secondary channel and RNAP active-center “i+1” site and. The SalBchlorohydrin can be altered with little loss of activity (Tables 1-2),and has unique chemical reactivity (Examples 6-7). Accordingly, it isinferred herein that it should be possible—by semi-synthesis or by totalsynthesis—to append at the SalB residue-9 chlorohydrin moiety chemicalfunctionality that makes favorable interactions with the RNAP secondarychannel or active-center “i+1” site, thereby increasing the potency ofRNAP inhibitory activity and potentially increasing the potency ofantibacterial activity.

By way of example, appending a sidechain that carries hydrogen-bondingfunctionality at the SalA residue-9 epoxide moiety or SalB residue-9chlorohydrin moiety, could allow for favorable hydrogen-bondedinteractions with polar residues on the floor of the RNAP secondarychannel (e.g., residues β678, β1105, β1106, β′731, and β′736 in RNAPfrom Escherichia coli, and residues corresponding to, and alignablewith, these residues in RNAP from other bacterial species).

By further way of example, appending a sidechain carrying negativelycharged functionality at the SalA residue-9 epoxide moiety or SalBresidue-9 chlorohydrin moiety could allow for favorable electrostaticinteractions with positively charged residues on the floor of the RNAPsecondary channel (e.g., residues β678, β1106, and β′731 in RNAP fromEscherichia coli, and residues corresponding to, and alignable with,these residues in RNAP from other bacterial species).

Example 2 Crystal Structure of RNAP in Complex with Sal Derivative

To confirm the binding position and binding orientation of Sal inferredfrom the crystal structure of RNAP-SalA, x-ray diffraction data andbromine anomalous scattering data were collected for crystals ofEscherichia coli RNAP holoenzyme soaked with the bromine-containing Salderivative Sal-Br (compound 3; crystal soaks, structure determination,and structure refinement performed essentially as described for SalA inExample 1). Sal-Br contained a residue-9 bromohydrin moiety analogous tothe residue-9 chlorohydrin moiety of SalB, and was prepared bysemi-synthesis from SalA, exploiting the chemical reactivity of the SalAresidue-9 epoxide (Example 3). Sal-Br exhibited essentially fullRNAP-inhibitory activity and antibacterial activity (Tables 1-2).

Electron density for Sal-Br from crystals of RNAP-Sal-Br complex matchedelectron density for SalA in the RNAP-SalA complex. Bromine anomalousdifference density showed a single peak (FIGS. 3A-B). The peak waslocated adjacent to the electron density for Sal-Br, in the positionpredicted for the bromine atom of the Sal-Br residue-9 bromohydrincarbon atom (FIGS. 3A-B). The results unequivocally define the SalA andSal-Br binding positions and binding orientations.

Example 3 Synthesis of Sal Derivatives Exploiting Reactivity of SalAEpoxide: Sal-Br (Compound 3)

SalA (1; 5 mg; 4.9 μmol; prepared as in Trischman et al., J. Am. Chem.Soc., 116:757, 1994; provided by William Fenical, Scripps Institution ofOceanography) was dissolved in 0.25 ml chloroform at 25° C. To thesolution was added 48% HBr (10 μl, 89 μmol; Aldrich). The reactionmixture was stirred 15 min at 25° C., and then quenched with 200 μl 50%sodium bicarbonate. The organic layer was separated, re-washed with 100μl water, and dried to a white solid. Products were purified usingsilica flash chromatography (0-10% methanol in chloroform as eluent).Yield: 5 mg, 93%. MS (MALDI): calculated: m/z 1099.41, 1101.41; found:1122.48, 1124.48 (M+Na⁺).

Example 4 Synthesis of Sal Derivatives Exploiting Reactivity of SalAEpoxide: Sal-OH (Compound 4)

SalA (1; 1 mg; 0.98 μmol; prepared as in Trischman et al., J. Am. Chem.Soc., 116:757, 1994; provided by William Fenical, Scripps Institution ofOceanography) was dissolved in 0.5 ml n-butanol, and 1 μl 98% sulfuricacid was added. The reaction mixture was heated 10 min at 100° C. in amicrowave reactor (Initiator, Biotage, Inc.), cooled to 25° C., and thenneutralized with 400 μl 50% sodium bicarbonate. The organic layer wasretrieved and evaporated to dryness. Products were purified byreversed-phase HPLC [Luna C18, 5μ, 100 A, 250 mm×4.6 mm (Phenomenex,Inc.); A, 60% methanol; B, 75% methanol; 0-15 min, 0% B; 15-30 min,0-100% B; flow rate 1 ml/min)]. Compound 4 eluted at 38 min. Yield: 80μg, 7.7%. MS (MALDI): calculated: m/z 1037.50, found: 1060.50 (M+Na⁺).

Example 5 Synthesis of Sal Derivatives Exploiting Reactivity of SalAEpoxide: Sal-OR 5.1. Sal-OBu A (Compound 5A) and Sal-OBu B (Compound 5B)

SalA (1; 1 mg; 0.98 μmol; prepared as in Trischman et al., J. Am. Chem.Soc., 116:757, 1994; provided by William Fenical, Scripps Institution ofOceanography) was dissolved in 0.5 ml n-butanol, and 1 μl 98% sulfuricacid was added. The reaction mixture was heated 10 min at 100° C. in amicrowave reactor (Initiator, Biotage, Inc.), cooled to 25° C., and thenneutralized with 400 μl 50% sodium bicarbonate. The organic layer wasretrieved and evaporated to dryness. Products were purified byreversed-phase HPLC [Luna C18, 5μ, 100 A, 250 mm×4.6 mm (Phenomenex,Inc.); A, 60% methanol; B, 75% methanol; 0-15 min, 0% B; 15-30 min,0-100% B; flow rate 1 ml/min)]. Compound 5A eluted at 56 min. Compound5B eluted at 53 min.

Compound 5A: Yield: 140 μg, 9%. MS (MALDI): calculated: m/z 1093.62;found: 1094.63, 1116.59 (M+Na⁺).

Compound 5B: Yield: 69 μg, 4.4%. MS (MALDI): calculated: m/z 1093.62;found: 1094.63, 1116.59 (M+Na⁺).

Example 6 Synthesis of Sal Derivatives Exploiting Reactivity of SaIAEpoxide: Sal-SR 6.1. Sal-SBu (Compound 6)

Compound 6 is prepared as described for compound 5A, except that 0.5 mlbenzene, 5 μmol lithium perchlorate, and 10 μmol butanethiol are used inplace of 0.5 ml n-butanol, and 1 μl 98% sulfuric acid.

Example 7 Synthesis of Sal Derivatives Exploiting Reactivity of SalBChlorohydrin: Sal—NHR 7.1. Sal-NH(CH₂)₃NHBoc (Compound 7)

SalB (2; 5 mg; 4.7 μmol; prepared as in Trischman et al., J. Am. Chem.Soc., 116:757, 1994; provided by William Fenical, Scripps Institution ofOceanography) was dissolved in 1 ml ethanol, andN-Boc-1,3-diaminopropane (3.4 mg, 19.5 μmol; Aldrich, Inc.) was added.The reaction mixture was heated 5 min at 150° C. in a microwave reactor(Initiator; Biotage, Inc.), cooled to 25° C., and then evaporated todryness. Products were purified by reversed-phase HPLC [Luna C18, 5μ,100 A, 250 mm×4.6 mm (Phenomenex, Inc.); A, 60% methanol; B, 75%methanol; 0-15 min, 0% B; 15-30 min, 0-100% B; flow rate 1 ml/min)].Compound 7 eluted at 35 min. Yield: 0.261 mg, 4.5%. MS (MALDI):calculated: m/z 1193.39; found: 1216.71 (M+Na⁺).

7.2. Sal-NH(CH₂)₃NHBoc (Compound 8)

Compound 8 is prepared from compound 7 by reaction with 50 μltrifluoroacetic acid in 200 μl chloroform for 30 min at 25° C., and ispurified by reversed-phase HPLC.

7.3. Sal-NH(CH₂)₆NHBoc (Compound 9)

SalB (2; 10 mg; 9.5 μmol; prepared as in Trischman et al., J. Am. Chem.Soc., 116:757, 1994; provided by William Fenical, Scripps Institution ofOceanography) was dissolved in 1 ml ethanol, and N-Boc-1,6-diaminohexane(4.1 mg, 18.95 μmol; Acros, Inc.) was added. The reaction mixture washeated 6 min at 160° C. in a microwave reactor (Initiator; Biotage,Inc.), cooled to 25° C., and then evaporated to dryness. Products werepurified by reversed-phase HPLC [Luna C18, 5μ, 100 A, 250 mm×4.6 mm(Phenomenex, Inc.); A, 60% methanol; B, 75% methanol; 0-15 min, 0% B;15-30 min, 0-100% B; flow rate 1 ml/min)]. Compound 8 eluted at 39 min.Yield: 1.46 mg, 14%. MS (MALDI): calculated: m/z 1235.67; found: 1236.56(M+H⁺), 1258.58 (M+Na⁺).

7.4. Sal-NH(CH₂)₆NHBoc (Compound 10)

Compound 10 is prepared from compound 9 by reaction with 50 μltrifluoroacetic acid in 200 μl chloroform for 30 min at 25° C. and ispurified by reversed-phase HPLC.

Example 8 RNAP-Inhibitory Activity

Radiochemical RNAP assays with Escherichia coli RNAP and Staphylococcusaureus RNAP were performed as follows: Reaction mixtures contained (10μl): 0-100 μM test compound, bacterial RNAP holoenzyme [75 nMEscherichia coli RNAP holoenzyme (prepared as in Mukhopadhyay et al.,Meths. Enzymol. 371:144-159, 2003) or 75 nM Staphylococcus aureus RNAPcore enzyme and 300 nM Staphylococcus aureus σ ^(A) (prepared as inSrivastava et al., Curr. Opin. Microbiol. 14:532-543, 2011)], 20 nM DNAfragment N25-lacUV5-14 [positions −100 to −1 of the bacteriophage T5 N25promoter followed by positions +1 to +29 of the lacUV5(+10A;+15C)promoter; prepared by PCR amplification of a syntheticnontemplate-strand oligodeoxyribonucleotide], 0.5 mM ApA, 100 μM□[α³²P]UTP (0.2 Bq/fmol), 100 μM ATP, and 100 μM GTP in TB (50 mMTris-HCl, pH 7.9, 100 mM KCl, 10 mM MgCl₂, 1 mM DTT, 100 μg/ml bovineserum albumin, and 5% glycerol). Reaction components except DNA, ApA,and NTPs were pre-incubated 10 min at 24° C.; DNA was added and reactionmixtures were incubated 10 min at 37° C.; ApA, 0.15 μl 7 μM [α³²P]UTP(200 Bq/fmol), ATP, and GTP were added and reaction mixtures wereincubated 5 min at 37° C.; and 0.5 μl 2 mM UTP was added and reactionmixtures were incubated 5 min at 37° C. Reactions were terminated byadding 10 μl A loading buffer (80% formamide, 10 mM EDTA, 0.02%bromophenol blue, and 0.02% xylene cyanol) and heating 2 min at 95° C.Products were applied to 7 M urea 15% polyacrylamide (19:1acrylamide:bisacrylamide) slab gels (Bio-Rad), electrophoresed in TBE(90 mM Tris-borate, pH 8.0, and 2 mM EDTA), and analyzed bystorage-phosphor scanning (Typhoon; GE Healthcare, Inc.).

Radiochemical assays with human RNAP I, II, and III were performedessentially as described [Sawadogo and Roeder, Cell 43:165-75, 1985].Reaction mixtures contained (20 μl): 0-100 μM test compound, 8 UHeLaScribe Nuclear Extract (Promega, Inc.), 1 μg human placental DNA(Sigma-Aldrich), 400 μM ATP, 400 μM [α³²P]UTP (0.11 Bq/fmol), 400 μMCTP, 400 μM GTP, 50 mM Tris-HCl, pH 8.0, 7 mM HEPES-NaOH, 70 mM(NH₄)₂SO₄, 50 mM KCl, 12 mM MgCl₂, 5 mM DTT, 0.1 mM EDTA, 0.08 mMphenylmethylsulfonyl fluoride, and 16% glycerol. Reaction componentsother than DNA and NTPs were pre-incubated 10 min at 30° C., DNA wasadded and reaction mixtures were incubated 15 min at 30° C., NTPs wereadded and reaction mixtures were incubated 60 min at 30° C. Reactionmixtures were spotted on DE81 filter discs (Whatman, Inc.; pre-wettedwith water) and incubated 1 min at room temperature. Filters were washedwith 3×3 ml Na₂HPO₄, 2×3 ml water, and 3 ml ethanol, using a filtermanifold (Hoefer, Inc.). Filters were placed in scintillation vialscontaining 10 ml Scintiverse BD Cocktail (Thermo Fisher, Inc.), andradioactivity was quantified by scintillation counting (LS6500;Beckman-Coulter, Inc.).

Fluorescence-detected RNAP assays with Escherichia coli RNAP wereperformed by a modification of the procedure of Kuhlman et al., Anal.Biochem. 324:183-190, 2004]. Reaction mixtures contained (20 μl): 0-100nM test compound, 75 nM Escherichia coli RNAP σ⁷⁰ holoenzyme, 20 nM 384bp DNA fragment containing the bacteriophage T4 N25 promoter, 100 μMATP, 100 μM GTP, 100 μM UTP, 100 μM CTP, 50 mM Tris-HCl, pH 8.0, 100 mMKCl, 10 mM MgCl₂, 1 mM DTT, 10 μg/ml bovine serum albumin, and 5.5%glycerol. Reaction components other than DNA and NTPs were pre-incubatedfor 10 min at 37° C. Reactions were carried out by addition of DNA andincubation for 5 min at 37° C., followed by addition of NTPs andincubation for 60 min at 37° C. DNA was removed by addition of 1 μl 5 mMCaCl₂ and 2 U DNaseI (Ambion, Inc.), followed by incubation for 90 minat 37° C. RNA was quantified by addition of 100 μl RiboGreen RNAQuantitation Reagent (Invitrogen, Inc.; 1:500 dilution in Tris-HCl, pH8.0, 1 mM EDTA), followed by incubation for 10 min at 25° C., followedby measurement of fluorescence intensity [excitation wavelength=485 nmand emission wavelength=535 nm; QuantaMaster QM1 spectrofluorometer(PTI, Inc.)].

Half-maximal inhibitory concentrations (IC50s) were calculated bynon-linear regression in SigmaPlot (SPSS, Inc.).

Example 9 Antibacterial Activity

Antibacterial activity was quantified using broth microdilution[Clinical and Laboratory Standards Institute (CLSI/NCCLS), Methods forDilution Antimicrobial Susceptibility Tests for Bacteria That GrowAerobically; Approved Standard, Eighth Edition. CLIS Document M07-A8(CLIS, Wayne Pa.), 2009]. Assays with Enterobacter cloacae ATCC13047,Pseudomonas aeruginosa ATCC 10145, and Escherichia coli D21f2toIC,employed a starting cell density of 2-5×10⁵ cfu/ml, Mueller Hinton IIcation adjusted broth (BD Biosciences, Inc.), and an air atmosphere.Assays with Haemophilus influenzae ATCC49247 and Neisseria gonorrhoeaeATCC19424 employed a starting cell density of 2-5×10⁵ cfu/ml,Haemophilus Test Medium broth (Barry et al., 1993) and a 5% CO2/95% airatmosphere. MIC50 was defined as the minimal concentration resulting in≧50% inhibition of bacterial growth.

Example 10 Cytotoxicity

MICs for mammalian cells (Vero E6) in culture were quantified usingCellTiter96 assay (Promega. Inc.; procedures as specified by themanufacturer).

Screening data for SalA and SalB (compounds 1 and 2) and representativecompounds of this invention (compounds 3-9) are presented in Tables 1-2:

TABLE 1 RNAP-inhibitory activity (fluorescent-detected RNAP assays) IC50IC50 Escherichia coli human RNAP RNAP I/II/III compound (μM) (μM) SalA(1) 1 >100 SalB (2) 1 >100 Sal-Br (3) 3 >100 Sal-OH (4) 2 Sal-OBu A (5A)6 Sal-OBu B (5B) >25 Sal-NH(CH₂)₃NHBoc (7) 0.6 Sal-NH(CH₂)₆NHBoc (9) 1

TABLE 2 Antibacterial activity MIC50 MIC50 Escherichia coli Enterobactercloacae D21f2toIC ATCC 13047 compound (μg/ml) (μg/ml) SalA (1) 0.0241.56 SalB (2) 0.098 6.25 Sal-Br (3) 0.049 1.56 Sal-OH (4) 0.78 25Sal-OBu A (5A) 1.56 12.5 Sal-NH(CH₂)₃NHBoc (7) 1.56 100Sal-NH(CH₂)₆NHBoc (9) 0.78 25

TABLE 3 Absence of cytotoxicity to mammalian cells in culture MIC50 VeroE6 ATCC CRL1586 compound (μg/ml) SalA (1) >100 SalB (2) >100 Sal-Br (3)>100

All documents cited herein are incorporated by reference. While certainembodiments of invention are described, and many details have been setforth for purposes of illustration, certain of the details can be variedwithout departing from the basic principles of the invention.

The use of the terms “a” and “an” and “the” and similar terms in thecontext of describing embodiments of invention are to be construed tocover both the singular and the plural, unless otherwise indicatedherein or clearly contradicted by context. The terms “comprising,”“having,” “including,” and “containing” are to be construed asopen-ended terms (i.e., meaning “including, but not limited to”) unlessotherwise noted. Recitation of ranges of values herein are merelyintended to serve as a shorthand method of referring individually toeach separate value falling within the range, unless otherwise indicatedherein, and each separate value is incorporated into the specificationas if it were individually recited herein. In addition to the orderdetailed herein, the methods described herein can be performed in anysuitable order unless otherwise indicated herein or otherwise clearlycontradicted by context. The use of any and all examples, or exemplarylanguage (e.g., “such as”) provided herein, is intended merely to betterilluminate embodiments of invention and does not necessarily impose alimitation on the scope of the invention unless otherwise specificallyrecited in the claims. No language in the specification should beconstrued as indicating that any non-claimed element is essential to thepractice of the invention.

1-24. (canceled)
 25. A method of synthesis of a compound of formula (I):

wherein: X is one of —Br, —I, —OR, —SR, and —NHR; and Y is OH; each R isindependently H or a branched or unbranched, saturated or unsaturated,hydrocarbon chain, having from 3 to 15 carbon atoms, wherein one or moreof the carbon atoms is optionally replaced by (—O—) or (—NR^(a)—), andwherein the chain is optionally substituted on carbon with one or moresubstituents independently selected from the group consisting of(C₁-C₆)alkoxy, (C₃-C₆)cycloalkyl, (C₁-C₆)alkanoyl, (C₁-C₆)alkanoyloxy,(C₁-C₆)alkoxycarbonyl, (C₁-C₆)alkylthio, azido, cyano, nitro, halo,hydroxy, oxo, carboxy, aryl, aryloxy, heteroaryl, heteroaryloxy, ahydrogen-bonding group, and a negatively charged functional group; andeach R^(a) is independently H or (C₁-C₆)alkyl; or a salt thereof,comprising reacting: a) salinamide A with HX in the presence of an acid;or b) salinamide A with HX in the presence of a base; or c) salinamide Awith Y′X, wherein Y′ is a cation; or d) salinamide B with HX in theoptional presence of a base; or e) salinamide B with Y′X, wherein Y′ isa cation.
 26. The method of claim 25, comprising reacting salinamide Awith HX in the presence of an acid.
 27. The method of claim 25,comprising reacting salinamide A with HX in the presence of a base. 28.The method of claim 25, comprising reacting salinamide A with Y′X,wherein Y′ is a cation.
 29. The method of claim 25, comprising reactingsalinamide B with HX in the optional presence of a base.
 30. The methodof claim 25, comprising reacting salinamide B with Y′X, wherein Y′ is acation.
 31. A method comprising deprotecting a compound:

to provide a corresponding deprotected amine:


32. A method comprising deprotecting a compound:

to provide a corresponding deprotected amine:


33. A compound selected from:


34. A method to inhibit a bacterial RNA polymerase comprising contactinga bacterial RNA polymerase with a compound of formula (I):

wherein: X is one of —Br, —I, —OR, —SR, and —NHR; Y is one of —Br, —I,—OR, —SR, and —NHR; and at least one of X and Y is OH; each R isindependently H or a branched or unbranched, saturated or unsaturated,hydrocarbon chain, having from 3 to 15 carbon atoms, wherein one or moreof the carbon atoms is optionally replaced by (—O—) or (—NR^(a)—), andwherein the chain is optionally substituted on carbon with one or moresubstituents independently selected from the group consisting of(C₁-C₆)alkoxy, (C₃-C₆)cycloalkyl, (C₁-C₆)alkanoyl, (C ₁-C₆)alkanoyloxy,(C₁-C₆)alkoxycarbonyl, (C₁-C₆)alkylthio, azido, cyano, nitro, halo,hydroxy, oxo, carboxy, aryl, aryloxy, heteroaryl, heteroaryloxy, ahydrogen-bonding group, and a negatively charged functional group; andeach R^(a) is independently H or (C₁-C₆)alkyl; or a salt thereof.
 35. Amethod to treat a bacterial infection in an animal comprisingadministering a compound of formula (I):

wherein: X is one of —Br, —I, —OR, —SR, and —NHR; Y is one of —Br, —I,—OR, —SR, and —NHR; and at least one of X and Y is OH; each R isindependently H or a branched or unbranched, saturated or unsaturated,hydrocarbon chain, having from 3 to 15 carbon atoms, wherein one or moreof the carbon atoms is optionally replaced by (—O—) or (—NR^(a)—), andwherein the chain is optionally substituted on carbon with one or moresubstituents independently selected from the group consisting of(C₁-C₆)alkoxy, (C₃-C₆)cycloalkyl, (C₁-C₆)alkanoyl, (C₁-C₆)alkanoyloxy,(C₁-C₆)alkoxycarbonyl, (C₁-C₆)alkylthio, azido, cyano, nitro, halo,hydroxy, oxo, carboxy, aryl, aryloxy, heteroaryl, heteroaryloxy, ahydrogen-bonding group, and a negatively charged functional group; andeach R^(a) is independently H or (C₁-C₆)alkyl; or a pharmaceuticallyacceptable salt thereof, to the animal.
 36. An assay for inhibition of aRNA polymerase comprising contacting a bacterial RNA polymerase with acompound of formula (I):

wherein: X is one of —Br, —I, —OR, —SR, and —NHR; Y is one of —Br, —I,—OR, —SR, and —NHR; and at least one of X and Y is OH; each R isindependently H or a branched or unbranched, saturated or unsaturated,hydrocarbon chain, having from 3 to 15 carbon atoms, wherein one or moreof the carbon atoms is optionally replaced by (—O—) or (—NR^(a)—), andwherein the chain is optionally substituted on carbon with one or moresubstituents independently selected from the group consisting of(C₁-C₆)alkoxy, (C₃-C₆)cycloalkyl, (C ₁ -C₆)alkanoyl, (C₁-C₆)alkanoyloxy, (C₁-C₆)alkoxycarbonyl, (C₁-C₆)alkylthio, azido, cyano,nitro, halo, hydroxy, oxo, carboxy, aryl, aryloxy, heteroaryl,heteroaryloxy, a hydrogen-bonding group, and a negatively chargedfunctional group; and each R^(a) is independently H or (C₁-C₆)alkyl; ora salt thereof.
 37. An assay for potential antibacterial activitycomprising contacting a bacterium with a compound of formula (I):

wherein: X is one of —Br, —I, —OR, —SR, and —NHR; Y is one of —Br, —I,—OR, —SR, and —NHR; and at least one of X and Y is OH; each R isindependently H or a branched or unbranched, saturated or unsaturated,hydrocarbon chain, having from 3 to 15 carbon atoms, wherein one or moreof the carbon atoms is optionally replaced by (—O—) or (—NR^(a)—), andwherein the chain is optionally substituted on carbon with one or moresubstituents independently selected from the group consisting of(C₁-C₆)alkoxy, (C₃-C₆)cycloalkyl, (C₁-C₆)alkanoyl, (C₁-C₆)alkanoyloxy,(C₁-C₆)alkoxycarbonyl, (C₁-C₆)alkylthio, azido, cyano, nitro, halo,hydroxy, oxo, carboxy, aryl, aryloxy, heteroaryl, heteroaryloxy, ahydrogen-bonding group, and a negatively charged functional group; andeach R^(a) is independently H or (C₁-C₆)alkyl; or a salt thereof.